M2: Oxphos - A Closer Look L15 Flashcards
What are the 2 major subunits of ATPase? Where is it located?
F1: hydrophilic and facing the matrix
Fo: In mitochondrial membrane and hydrophobic
Describe the F1 component of ATP synthase.
- 3 alpha and 3 beta subunits arranged in a cyclical way.
- The beta subunit is responsible for catalysis of ATP synthesis. At any given time point, only one B subunit is making an ATP. (One beta will be empty, one bound to ADP the other is bound to ATP).
Describe the Fo component of ATP synthase.
Fo is a transmembrane ring with 8 subunits that rotates. The whole rotor rotates as a single body. The # of protons that are translocated for every ATP synthesized is fixed. This is determined by the number of subunits = 8/3ATP.
How do you calculate how many protons are translocated per ATP from ATP synthase?
Since 3 catalytic sites are present on F1, the number of protons translocated/ATP is equal to the n umber of C subunits divided by 3. 8/3=2.7.
What are the 3 states of the beta subunits of the F1.
O= open, nothing bound L= loose, binds ligands ADP and Pi. Inactive T= tight, catalytically active and synthesizes ATP
Describe the reaction sequence of the beta subunits starting with ATP in the T subunit.
- L and O beta subunits are unbound. T has ATP.
- L binds ADP and Pi. T still has ATP. O is still empty
- An energy dependent conformational change occurs:
- L to T causing the synthesis of ATP.
- T to O causing release of ATP that was in T
- O to L stays empty (now can bind new ADP and Pi)
RESTART
Describe the F1-Fo ATPase as a rotary engine.
The rotor is the c ring with 8 subunits. The C ring and the gamma and epsilon subunits is the rotor (what rotates). The stator (stays stationary) is alpha and beta (F1), delta, b2 and a. (see L15 S9)
The rotation of the rotor occurs due to migrations of protons from inter membrane space into the matrix.
Protons from the inter membrane space enter the channel of the c ring and bind a c subunit. This causes a conf. change of the c subunit which pushes it against a previous one and causes it to rotate counter clockwise. As it rotates, the next empty site can be bound by another proton. It keeps going around until it reaches the second hydrophilic channel and the proton is released into the matrix. The C subunit can now take a new proton.
How was the rotation of the c ring of ATP synthase experimentally confirmed?
A scientist made the ATP synthase stationary by binding it to a his tag bound to a nickel surface. The C-ring is facing up. He attached an actin filament to the c ring. He observed the actin filament turning. As the c ring rotated counter clockwise.
Explain the experiment that showed how mitochondrial uncoupling can reduce obesity.
Mice fed a high fat diet over 8 weeks. Some mice were fed a high fat diet alone, some were fed a high fat diet and given DNP uncoupler.
The DNP mice gain less weight even though they’re eating the same food. So if they’re losing mass even tho eating same food there must be an increase in energy expenditure.
Also, it was seen that the difference in mass came from the loss of fat mass.
The consumption of oxygen in animals was also observed: you can see that as you increase the DNP dose, there is an increase in metabolism AKA oxygen consumption (energy expenditure).
A change in body weight was also observed: As you increase DNP, there is an increase in energy expenditure, and an increase in weight loss.
How is human brown adipose tissue correlated with obesity?
Human BAT is inversely correlated with obesity. Individuals with less body fat have more brown adipose tissue activity. Why? the endogenous uncoupler UCP1 is located in brown adipose tissue.
Explain how UCP1 works in the mitochondria of brown adipose tissue.
Uncoupling protein 1 (UCP1) is like the endogenous DNP. It allows protons to renter the matrix which bypasses ATP synthase. UCP1 is not always active, it needs to be turned on. It is regulated by norepinephrine. Brown adipocytes express adrenergic receptors where norepinephrine binds, it signals to activate adenylate cyclase, which produces the second messenger cAMP. cAMP activates PKA through allosteric regulation, PKA then phosphorylates hormone-sensitive triacylglycerol lipase. When it is phosphorylated, it catalyzes the release of fatty acids from stored fat within fat cells. The fatty acids can go in the mitochondria for beta oxidation (they fuel the CAC) and they bind UCP1 and activate it.
